Islet Damage Research Articles

Injury factors alter miRNAs profiles of exosomes derived from islets and circulation.

Islets damage is a major abnormality underling diabetes. Recent studies suggested the value of exosomes in diagnosis. This study aimed to investigate the impact of injury factors on the miRNA profiles of islet exosomes and determine whether circulating exosomal miRNAs is suitable as biomarkers of islets damage. Islets were isolated from ICR mice and induced injury in vitro by mixed cytokines (Tumor Necrosis Factor-α, Interleukin -1β and Interferon-γ) or streptozotocin (STZ), and exosomes were derived from the cultural supernatant. Using miRNA microarray analysis, we found 22 and 11 differentially expressed miRNAs in islet exosomes of STZ and cytokines treatment, respectively, including 6 miRNAs as the intersection of two injured conditions. Thereinto, mmu-miR-375-3p and mmu-miR-129-5p could be validated by qRT-PCR. Then, Serum exosomes were isolated from STZ injected mice and subjects with various glucose metabolism states and diabetic duration. qRT-PCR demonstrated exosomal mmu-miR-375-3p dramatically increased in serum of STZ treated mouse prior to the disturbance of blood glucose and insulin. In human serum exosomes, hsa-miR-375-3p was elevated in new-onset diabetes patients. Overall, our results suggest that injury factors changed miRNA profiles of exosomes derived from islets and exosomal miR-375-3p showed promising potential as a biomarker of islets damage.

Polysaccharides from Mushroom Fruiting Bodies Improve Insulin Resistance by Inhibiting Lipid Deposition Via Autophagy Enhancement

Insulin resistance is the main cause of type 2 diabetes. Polysaccharide is one of the main active components in mushrooms and has been reported that certain mushroom polysaccharides have beneficial effects against type 2 diabetes. However, the structural features and mechanisms of polysaccharides with hypoglycemic activity have not been elucidated. In this paper, six alkali-soluble total polysaccharides, six neutral polysaccharides and six acidic polysaccharides were extracted and purified from mushrooms fruiting bodies of different species. The effects of these polysaccharides on improving insulin resistance were compared using high fatty acids and glucose-induced hepatocytes. Among them, the neutral polysaccharide AAMP-N of Armillaria mellea fruiting body showed the most significant activity, suggesting that galactoglucan is the main active domain. AAMP-N inhibited intracellular fat accumulation and enhanced insulin sensitivity by activating autophagy. Autophagy is an essential lysosomal degradation pathway implicated in the development of type 2 diabetes. Consistent with the in vitro results, AAMP-N enhanced hepatic autophagy, lowered blood glucose, and modulated lipid metabolism in db/db mice. In addition, AAMP-N also protected damaged pancreatic islets in mice. Our results demonstrated for the first time a role of natural polysaccharides from mushrooms in the improvement of insulin sensitivity and metabolism, and provided basis for the development of mushroom polysaccharides as hypoglycemic healthy food. Funding Statement: This work was supported by National Natural Science Foundation of China (No. 31872674), Jilin Province Pharmaceutical Industry Development Fund (No. 20180311068YY), Jilin Province Development and Reform Commission (2018C047-2), and Fundamental Research Funds for the Central Universities (No. 2412017FZ018). Declaration of Interests: The authors have declared no conflicts of interest. Ethics Approval Statement: All experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals of Northeast Normal University.

Protective Role of Melatonin in Streptozotocin Induced Pancreatic Damages in Diabetic Wistar Rat

Hyperglycemia is a representative hallmark and risk factor for diabetes and is closely linked to diabetes associated complications. The aim of the present study was to evaluate the therapeutic potential of exogenous melatonin against the streptozotocin induced pancreatic damages in rats. Streptozotocin was injected for consecutive 6 days. Diabetes was confirmed by blood glucose measurement after 72 h and on 7th day after injection. Animals having blood glucose level above 250 mg dL-1 were considered as diabetic and were administered exogenous melatonin for 4 weeks. Animals were euthanized after last dose, pancreas were dissected out, weighed and fixed in Bouin's fixative for histology and further tissues were kept at -20°C for biochemistry. Diabetic rats displayed significant increase in lipid peroxidation, but pancreatic weight index, antioxidant system (GSH, SOD and CAT) showed decrease. Melatonin treatment to diabetic rats restored the alteration in physiological and biochemical markers. Results were supported by the histopathological observations, STZ treated pancreas showed damage in islets of langerhans, while as melatonin treated diabetic rats recovered the cellular architecture which inturn normalize the function of the pancreas. Therefore, melatonin might be considered as a molecule to protect the pancreatic damages.

The methanolic extract of Thymus praecox subsp. skorpilii var. skorpilii restores glucose homeostasis, ameliorates insulin resistance and improves pancreatic β-cell function on streptozotocin/nicotinamide-induced type 2 diabetic rats

Ethnopharmacological relevanceThymus praecox subsp. skorpilii var. skorpilii (syn. Thymus praecox subsp. jankae (Celak.) Jalas) is consumed as a Turkish folk medicine for the treatment of spasm, sore throat and shortness of breath, also having strong antioxidant activity and the leaves of the plant have been utilized for the treatment of diabetes as the decoction in Turkey. Aim of the studyIn the present study, we aimed to investigate the potential mechanism of antidiabetic action of Thymus praecox subsp. skorpilii var. skorpilii methanolic extract (TPSE) on streptozotocin (STZ)/nicotinamide (NA)-induced type 2 diabetic rats. Materials and methodsSprague Dawley rats were randomly divided into four groups; control, diabetes, TPSE (100 mg/kg b.w, p.o.) and metformin group (400 mg/kg b.w, p.o.). Diabetes was established in all groups except control group by 55 mg/kg STZ (i.p.) for once 15 min after 100 mg/kg NA injection. 3 days after STZ/NA injection, treatments were administered for three weeks and then rats were decapitated; tissue and blood samples were obtained for measuring the level of glucose transporters (both GLUTs and sodium glucose co-transporters (SGLTs)), enzymes related to glucose (Hexokinase (HK), phosphoenolpyruvate carboxykinase (PEPCK), α-glucosidase) and lipid metabolism (Acetyl-coenzyme carboxylase (ACC)), AST, ALT, creatinine, insulin, anti-inflammatory (IL-10) and inflammatory (TNF-α, IL-1β, IL-6) cytokines, AMP-activated protein kinase (AMPK), peroxisome proliferator-activated receptor gamma (PPAR-γ) and glucagon like peptide-1 (GLP-1). Histopathological alterations of the pancreas were examined. ResultsAfter three weeks of treatment, TPSE has exhibited a significant reduction of plasma levels of the proinflammatory cytokines. Besides, TPSE treatment elevated plasma insulin levels and normalized blood glucose levels. Moreover, it improved the values of AMPK in liver and GLP-1 in pancreas. Increased α-glucosidase, PEPCK, GLUT-2 and SGLTs levels with the induction of diabetes considerably lowered with TPSE treatment. Especially on SGLT-2, TPSE achieved a more prominent decrease. After the atrophy in Langerhans islets due to diabetes induction, treatment was found to prevent the damage of islets. ConclusionsBased on the findings presented here, it has been concluded that TPSE has marked antidiabetic effects through various pathways on STZ/NA-induced diabetic rats and it may potentially be used as an effective treatment for type 2 diabetes mellitus (T2DM). Further research on isolation of the bioactive components is underway.

Extension of the mutation spectrum of PAX6 from three Chinese congenital aniridia families and identification of male gonadal mosaicism.

BackgroundCongenital aniridia is a severe autosomal dominant binocular developmental disorder, the primary feature of which is congenital absence or hypoplasia of the iris. PAX6 is the main disease‐causing gene of congenital aniridia; inheritance is autosomal dominant. But the current mutations do not fully explain this disorder.MethodsWe investigated the mutation profile of genes related in three Chinese families with congenital aniridia through targeted sequencing technology. And we validated the candidate variants by PCR‐based Sanger sequencing. Different degree impairments of islet function were observed in the patients with aniridia by carbohydrate tolerance butter and insulin release tests in our study.ResultsWe identified four novel mutations of PAX6 from three Chinese families with congenital aniridia, which included heterozygous double mutation c.879_880delCA (p.S294Cfs*46) and c.1124C>G (p.P375R) in Family 1 with three patients, heterozygous frameshift mutation c.308delG (p.P103Qfs*21) in Family 2 with one patient, and c.1192delT (p.S398Pfs*126) in Family 3 with two patients. The three frameshift mutations of PAX6 are co‐segregated with the aniridia from controls in the families, but the novel missense mutation is not co‐segregated with the phenotype. The frameshift mutations in Family 1 and Family 2 have effects to truncate the protein, but the frameshift mutation in Family 3 will prolong it. We confirmed the phenomenon of male gonadal mosaicism of PAX6 by the sequencing of two linked novel mutations in Family 1. Most of the patients with isolated aniridia have different degrees of islet damage through related clinical tests.ConclusionIt is therefore noteworthy that we found different types of pathogenic mutation, which have effects of truncating or prolonging protein leaded by frameshift mutation. Our results of this study extended the pathogenic mutation spectrum of PAX6 for congenital aniridia and demonstrated the male germline chimerism by molecular experiments.

Study of 2009 H1N1 Pandemic Influenza Virus as a Possible Causative Agent of Diabetes.

Recent studies have suggested that influenza A virus (IAV) might be involved in the etiology of diabetes. To address this question, we tested the ability of H1N1 pandemic IAV to infect, replicate, and damage human β cells/pancreatic islets in vitro and induce pancreatic damage and/or glucose metabolism alterations in chemical and autoimmune models of β cell damage in vivo. Moreover, we looked for direct and/or indirect evidence of correlation between IAV infection and autoimmunity/diabetes in humans. Human H1N1 A/California/2009-derived viruses infected human pancreatic islets in vitro, inducing a proinflammatory response associated with substantial increases of CXCL9 and CXCL10 release. In vivo, infected mice showed a clear susceptibility to the virus, with its localization also found in extrapulmonary organs, including the pancreas. Infection was able to induce mild modifications of glycemia in C57B6 mice after chemical damage of islets but did not modulate the autoimmune damage of islets in NOD mice. One of 69 nasopharyngeal swabs collected from patients at the onset of type 1 diabetes yielded positive results for IAV. Pancreas sections from 17 organ donors available from the Network for Pancreatic Organ Donors With Diabetes showed the persistence of CXCL10-positive cells in islet autoimmunity-positive subjects; however, extremely rare cells stained for viral RNA and not preferentially in autoimmune subjects. Influenza H1N1 pdm strains are able to infect and replicate in mammalian pancreatic cells both in vitro and in vivo but did not cause any functional impairment consistent with diabetes.

Sodium butyrate mitigates type 2 diabetes by inhibiting PERK-CHOP pathway of endoplasmic reticulum stress

Sodium butyrate (NaB), a histone deacetylase (HDAC) inhibitors, has been reported to attenuate hyperglycemia in rats. Our objective was to explore the effect and underlying mechanism of NaB on islet β-cell dysfunction and apoptosis in type 2 diabetic (T2DM) rats. T2DM models were induced by the combination of streptozotocin (STZ, 40 mg/kg) and high-fat-diet, while NaB (500 mg/kg/d) was intraperitoneally injected for 6 weeks in experimental groups. Our results suggested NaB mitigated hyperglycemia, lowered the levels of serum cholestenone (TC) and low-density lipoprotein (LDL-c), prevented body weight loss, and enhanced insulin resistance and glucose tolerance. NaB also improved diabetes-induced histological alteration of islet and functional damage; moreover, results of TUNEL and western blotting indicated NaB alleviated β-cell apoptosis. Further research showed NaB down-regulated the expression of endoplasmic reticulum stress (ERS) related proteins, including phosphorylated type I transmembrane ER-resident protein kinase (p-PERK), phosphorylated eukaryotic initiation factor 2α (p-eIF2α), activating transcription factor (ATF4) and CCAAT/enhancer-binding protein homologous protein (CHOP). Consequently, NaB mitigates type 2 diabetes by inhibiting PERK-CHOP pathway of ERS.

Effects of Buchholzia coriacea Seed on Nutrient Utilization and Serum Biochemical Parameters in Alloxan-Induced Diabetic Rat

The effects of Buchholzia coriacea seed on the nutrient utilization and biochemical parameters in alloxan-induced diabetic rat were evaluated. Buchholzia coriacea (BC) seed was washed, sliced, dried, pulverized and mixed with standard ration at 2.5%, 5%, 10% and 20%. The proximate nutrient composition of the standard and prepared rations was determined. The Buchholzia coriacea incorporated rations and standard ration were fed to alloxan-induced diabetic rats for 70 consecutive days. The nutrient utilization and biochemical parameters as well as the histopathology of pancreas of the treated rats were evaluated. The Buchholzia coriacea at 2.5% inclusion rate significantly (p < 0.05) improved the nutrient utilization and biochemical parameters that were compromised in diabetic rats fed with standard ration alone. The B. coriacea also reversed the pancreatic islet damage induced by alloxan. Buchholzia coriacea have potent antidiabetic and hypolipidemic activities and should not be incorporation in excess of 5% in the diet.

Early TLR4 Blockade Attenuates Sterile Inflammation-mediated Stress in Islets During Isolation and Promotes Successful Transplant Outcomes.

During the isolation process, pancreatic islets are exposed to an environment of sterile inflammation resulting in an upregulated inflammatory state before transplantation. Toll-like receptor 4 (TLR4) has been identified as a major mediator of sterile inflammation. Therefore, we sought to determine whether early TLR4 blockade would be effective in reducing the inflammatory burden in islets pretransplant. Islets from C57BL/6 mice were treated with a TLR4 antagonist during the pancreatic ductal perfusion and digestion steps of the isolation process. Islets were then analyzed for inflammation by RT-PCR and Western blot, and for viability and function in vitro. A syngeneic transplant model using a marginal mass of islets transplanted intraportally into mice with streptozotocin-induced diabetes was used to study transplant outcomes after early TLR4 blockade. Diabetic mice receiving 150 islets treated with early TLR4 blockade achieved euglycemia at a higher rate than mice receiving untreated islets (75% vs 29%; P < 0.05) and had improved long-term function (P < 0.05). Serum markers for islet damage and inflammation were significantly reduced posttransplant (P < 0.05). Both the expression of key inflammatory genes and the activation of mitogen-activated protein kinases were reduced by early TLR4 blockade. Islet viability was improved (P < 0.05) while preserving islet insulin secretory capacity postisolation. Early TLR4 blockade protects islets from sterile inflammation-mediated stress sustained during isolation and promotes positive transplant outcomes. Our findings support the use of early TLR4 blockade during clinical islet isolation procedures to reduce pretransplant inflammation and improve transplant outcomes.

NLRP3 inflammasome mediates oxidative stress-induced pancreatic islet dysfunction.

Inflammasomes are multiprotein inflammatory platforms that induce caspase-1 activation and subsequently interleukin (IL)-1β and IL-18 processing. The NLRP3 inflammasome is activated by different forms of oxidative stress, and, based on the central role of IL-1β in the destruction of pancreatic islets, it could be related to the development of diabetes. We therefore investigated responses in wild-type C57Bl/6 (WT) mice, NLRP3-/- mice, and mice deficient in apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC) after exposing islets to short-term hypoxia or alloxan-induced islet damage. NLRP3-deficient islets compared with WT islets had preserved function ex vivo and were protected against hypoxia-induced cell death. Furthermore, NLRP3 and ASC-deficient mice were protected against oxidative stress-induced diabetes caused by repetitive low-dose alloxan administration, and this was associated with reduced β-cell death and reduced macrophage infiltration. This suggests that the beneficial effect of NLRP3 inflammasome deficiency on oxidative stress-mediated β-cell damage could involve reduced macrophage infiltration and activation. To support the role of macrophage activation in alloxan-induced diabetes, we injected WT mice with liposomal clodronate, which causes macrophage depletion before induction of a diabetic phenotype by alloxan treatment, resulting in improved glucose homeostasis in WT mice. We show here that the NLRP3 inflammasome acts as a mediator of hypoxia and oxidative stress in insulin-producing cells, suggesting that inhibition of the NLRP3 inflammasome could have beneficial effects on β-cell preservation.

Phenolic Compounds from the Aerial Parts of Malva verticillata and their Anti-diabetic Effect

The EtOAc, n-BuOH, and aqueous fractions from the aerial parts of Malva verticillata have been shown to promote significant recovery from alloxan-induced pancreatic islet (PI) damage in zebrafish larvae at 10 μg/mL. Therefore, this study aimed to identify the principal active components of these plant parts and their pharmacological properties. Repeated SiO2 and octadecyl SiO2 column chromatography with the aerial parts of M. verticillata led to isolation of four phenolic compounds; these compounds were identified as benzyl-α-D-galactopyranoside (1), (-)-secoisolariciresinol-9'-O-β-D-glucopyranoside (2), transferulic acid (3), and trans-ferulic acid methyl ester (4) on the basis of physicochemical and spectroscopic analyses including infrared, nuclear magnetic resonance, and fast atom bombardment-mass spectroscopy. Compounds 14 were first isolated from M. verticillata in this study. Furthermore, compounds 1–4 recovered alloxan-induced PI damage in zebrafish. Especially, compound 3 recovered the size of the injured PIs by 83.8% ( p=0.0007) compared to the alloxan-induced group, while compound 4 by 33.4% ( p=0.0072). It is the first report that trans-ferulic acid (3) exhibited the protective effect on zebrafish larvae PIs damaged by alloxan.

Improved outcomes of islet autotransplant after total pancreatectomy by combined blockade of IL-1β and TNFα.

The efficacy of islet transplant is compromised by a significant loss of islet mass posttransplant due to an innate inflammatory reaction. We report the use of a combination of etanercept and anakinra (ANA+ETA) to block inflammatory islet damage in 100 patients undergoing total pancreatectomy with islet autotransplant. The patients were divided into 3 groups: no treatment (control [CTL]), etanercept alone (ETA), or a combination of etanercept and anakinra (ANA+ETA). Peritransplant serum samples were analyzed for protein markers of islet damage and for inflammatory cytokines. Graft function was assessed by fasting blood glucose, basal C-peptide, secretory unit of islet transplant objects (SUITO) index, and hemoglobin A1c . Administration of both antiinflammatory drugs was well tolerated without any major adverse events. Reductions in interleukin-6, interleukin-8, and monocyte chemoattractant protein 1 were observed in patients receiving ANA+ETA compared with the CTL group, while also showing a modest improvement in islet function as assessed by basal C-peptide, glucose, hemoglobin A1c , and SUITO index but without differences in insulin dose. These results suggest that double cytokine blockade (ANA+ETA) reduces peritransplant islet damage due to nonspecific inflammation and may represent a promising strategy to improve islet engraftment, leading to better transplant outcomes.

English

Several studies highlight beneficial effect of glabridin as an anti-hyperglycemic agent. However, the protective mechanisms of glabridin on improvement of pancreatic islets damage due to streptozotocin (STZ) exposure have not been adequately investigated. In the present study, we aimed to elucidate the effects of glabridin on restoration of pancreatic islet architecture, at least in part by restoring collagen I and fibronectin, through an activation of protein kinase Cs (PKCs) and transforming growth factor (TGF)-&beta;1 in STZ-induced pancreatic &beta;-cell injuries. Glabridin (40 mg/kg b.wt./day) was administrated to STZ-exposed rats for 8 weeks. The effects of glabridin were then investigated by measuring body weight, blood glucose level, pancreatic morphology, and protein expressions of PKCs, TGF-&beta;1, collagen I, and fibronectin. The results revealed that glabridin prevented weight loss and significantly decreased blood glucose level. Physical distortion and impairment with irregular outline was clearly observed in untreated STZ islets. Nevertheless, rat islets showed nearly normal architecture with a high &beta;-cell number after glabridin treatment. Glabridin also ameliorated the expression levels of PKCs, TGF-&beta;1, collagen I, and fibronectin in STZ-rat pancreas. These results provide novel evidence that glabridin significantly reduced pancreatic islets abnormalities in STZ-exposed rats and these effects could be associated with its hypoglycemic activity and with restoration of normal levels of PKCs, TGF-&beta;1, collagen I and fibronectin, which could subsequently contribute to maintaining islet structure and function. Key words: Glabridin, pancreas, diabetes mellitus, anti-hyperglycemia, anti-oxidant activity, streptozotocin.

Injury Factors Alter miRNAs Profiles of Exosomes Derived from Islets and Circulation

Islets damage is a major abnormality underling diabetes, thus timely monitoring islets destruction is critically needed. Recent studies discovered the biological functions of exosomes and suggested the value of exosomes in disease diagnosis and prognosis. This study aimed to investigate the impact of injury factors on the miRNA profiles of islet exosomes and determine whether circulating exosomal miRNAs would be suitable as biomarkers of islets cell damage. Islets were isolated from ICR mice and induced injury in vitro by mixed cytokines (TNFα, IL-1β and IFNγ) and streptozotocin (STZ) and exosomes were derived from the cultural supernatant. Using miRNA microarray analysis, we found 22 and 11 differentially expressed miRNAs in islet exosomes of STZ and cytokines treatment, respectively. 6 differentially expressed exosaml miRNAs were found both in STZ and cytokines treated group, and miR-375-3p and miR-129-5p were validated by quantitative RT-PCR. Serum exosomes were isolated from ICR mice of islets damage in vivo induced by STZ injection and subjects with various glucose metabolism states and diabetic duration. Quantitative RT-PCR demonstrated exosomal miR-375-3p dramatically increased in serum of STZ treated mouse prior to the turbulence of blood glucose and serum insulin. In human serum exosomes, miR-375-3p was elevated in newly new-onset diabetes patients. Overall, our results suggest that injury factors cytokines and STZ affected the miRNA profiles of exosomes derived from islets and exosomal miR-375-3p showed promising potential as a biomarker of islets damage. Further studies were necessary to purify islet-specific exosomes from circulation for improving the accuracy. Disclosure Q. Fu: None. H. Jiang: None. T. Yang: None.

Sensory Innervation of the Pancreatic Islet

Sensory innervation of viscera is an important component for proper maintenance of body homeostasis. Visceral stimuli are detected by free nerve endings of the vagus nerve and are transmitted to the hindbrain via sensory neurons. However, the signals that activate vagal sensory neurons in the periphery are not known. The objective of this study is to identify and characterize the population of vagal sensory neurons that innervates the pancreas. Using immunohistochemistry for sensory neuronal marker substance P, we show that sensory innervation targets islet periphery. Upon islet damage (in NOD model or following streptozotocin (STZ) treatment), density of islet sensory innervation increases. Expression of the immediate early gene c-Fos, shows that hindbrain neurons in the nucleus of the solitary tract (NTS) respond to severe islet inflammation induced by STZ. We assessed response profile of pancreatic sensory neurons using in-vivo calcium imaging of mouse vagal sensory ganglion (nodose). This method allows us to monitor in real time the response profile of sensory neurons and identify pancreas-specific subpopulations by chemical stimulation of the exteriorized pancreas. We have identified sensory neurons that respond to pancreatic application of serotonin (5HT) and cholecystokinin (CCK). The observed response profile suggests that these neuronal populations are tuned to sense differentially endocrine and exocrine pancreas. While endocrine sensory neurons are sensing local 5HT that is co-released with insulin, exocrine neurons sense CCK a hormone that stimulates release of digestive enzymes. Altogether our data suggest that pancreatic sensory neurons could be important regulators of pancreatic physiology. By taking a snapshot of endocrine and exocrine pancreatic microenvironments and sending this information to the brain, sensory neurons might trigger feedback loops and help the organ to be tuned to physiological needs. Disclosure M. Makhmutova: None. J. Weitz: None. R. Rodriguez Diaz: None. J. Almaca: None. E. Bernal-Mizrachi: None. A. Caicedo: None.

Development of Perfluorodecalin Contained Microcapsules for Islet Survival and Function

Hypoxic injury of islets is a major obstacle for encapsulated islet transplantation into the peritoneal cavity. To improve oxygen delivery to encapsulated islets, we integrated 20% of the oxygen carrier material, perfluorodecalin (PFD) contained alginate capsules with islets (PFD-alginate). Integration of PFD clearly improved islet viability and decreased reactive oxygen species production compared with islets encapsulated with alginate only (alginate) and naked islets exposed to hypoxia in vitro. In PFD-alginate capsules, HIF-1α expression was minimal, also insulin expression was well secreted. Furthermore, the best islet function represented by glucose-stimulated insulin secretion was observed for the PFD-alginate capsules. In vivo study, the marginal number naked islets and encapsulated islets (alginate and PFD-alginate) were transplanted into streptozotocin-induced diabetic mice. Non-fasting blood glucose levels and intraperitoneal glucose tolerance tests in the PFD-alginate group was lower than in the alginate group. The harvested islets stained positive for insulin in all groups, but the ratio of dead cell area was 4 times higher in the alginate group was than in the PFD-alginate group. In conclusion, PFD contained alginate microcapsules improved islet function and survival by minimizing the hypoxic damage of islets after intraperitoneal transplantation. Disclosure J. Kim: None. H. Park: None. K. Yoon: None. E. Lee: None.

IMG-1 Activates and Mobilizes Insulin-Producing Islet Progenitor Cells (CD133+/Insulin+) In Vitro

IMG-1 is a novel therapeutic for the treatment of types 1 and 2 diabetes that may have islet regenerative properties. 300 islets isolated from mouse pancreases were cultured in standard media with IMG-1 (0.5µg/mL) and compared with control non-treatment. Within 24 hours, the IMG-1 treated islets became smaller (less clustered) and islet cells appeared to actively migrate away from the clusters. Control islets maintained their clustered morphology and did not show any evidence of cell migration. Three days after IMG-1 treatment, there were both free-floating and attached cells in the culture. The free-floating cells were 30% viable and the attached cells and leftover islets were 85% viable. In contrast, control islet cultures had neither free-floating nor attached cells although islet clusters looked healthy (cell viability &amp;gt;90%). By day 10 of IMG-1 treatment, islet clusters were few in numbers (&amp;lt;10%), no free-floating cells remained, and the attached cells appeared to form colonies. Total cell viability was &amp;gt;90%. Control cells still had no migration or attachment, although within the islet clusters cell viability remained around &amp;gt;90%. At 25 days, islet cells from both cultures were fixed and stained for the progenitor cell marker, CD133, and insulin. IMG-1 treated colonies were 75% insulin-positive by immunohistostaining. Furthermore, these cells were 70% CD133-positive with 60% of the culture being CD133/Insulin double-positive. In contrast, non-treated islets were 90% insulin+ cells, but &amp;lt;5% CD133+ and &amp;lt;5% double positive (CD133/insulin). IMG-1 has a novel effect on islets and induces the activation and migration of islet cells out of the islet. These cells form colonies of CD133/insulin double-positive cells that putatively represent activated beta-cell progenitor cells that can be used to regenerate damaged islets. Disclosure R. Bottino: None. J.B. Pollett: Employee; Self; Imagine Pharma. N. Thai: Board Member; Self; Imagine Pharma. M. Trucco: None.

Kill two birds with one stone: making multi-transgenic pre-diabetes mouse models through insulin resistance and pancreatic apoptosis pathogenesis.

BackgroundType 2 diabetes is characterized by insulin resistance accompanied by defective insulin secretion. Transgenic mouse models play an important role in medical research. However, single transgenic mouse models may not mimic the complex phenotypes of most cases of type 2 diabetes.MethodsFocusing on genes related to pancreatic islet damage, peripheral insulin resistance and related environmental inducing factors, we generated single-transgenic (C/EBP homology protein, CHOP) mice (CHOP mice), dual-transgenic (human islet amyloid polypeptide, hIAPP; CHOP) mice (hIAPP-CHOP mice) and triple-transgenic (11β-hydroxysteroid dehydrogenase type 1, 11β-HSD1; hIAPP; CHOP) mice (11β-HSD1-hIAPP- CHOP mice). The latter two types of transgenic (Tg) animals were induced with high-fat high-sucrose diets (HFHSD). We analyzed the diabetes-related symptoms and histology features of the transgenic animals.ResultsComparing symptoms on the spot-checked points, we determined that the triple-transgene mice were more suitable for systematic study. The results of intraperitoneal glucose tolerance tests (IPGTT) of triple-transgene animals began to change 60 days after induction (p < 0.001). After 190 days of induction, the body weights (p < 0.01) and plasma glucose of the animals in Tg were higher than those of the animals in Negative Control (Nc). After sacrificed, large amounts of lipid were found deposited in adipose (p < 0.01) and ectopically deposited in the non-adipose tissues (p < 0.05 or 0.01) of the animals in the Tg HFHSD group. The weights of kidneys and hearts of Tg animals were significantly increased (p < 0.01). Serum C peptide (C-P) was decreased due to Tg effects, and insulin levels were increased due to the effects of the HFHSD in the Tg HFHSD group, indicating that damaged insulin secretion and insulin resistance hyperinsulinemia existed simultaneously in these animals. The serum corticosterone of Tg was slightly higher than those of Nc due to the effects of the 11βHSD-1 transgene and obesity. In Tg HFHSD, hepatic adipose deposition was more severe and the pancreatic islet area was enlarged under compensation, accompanying apoptosis. In the transgenic control diet (Tg ControlD) group, hepatic adipose deposition was also severe, pancreatic islets were damaged, and their areas were decreased (p < 0.05), and apoptosis of pancreatic cells occurred. Taken together, these data show the transgenes led to early-stage pathological changes characteristic of type 2 diabetes in the triple-transgene HFHSD group. The disease of triple-transgenic mice was more severe than that of dual or single-transgenic mice.ConclusionThe use of multi-transgenes involved in insulin resistance and pancreatic apoptosis is a better way to generate polygene-related early-stage diabetes models.

Improvement of islet function and survival by integration of perfluorodecalin into microcapsules in vivo and in vitro.

Hypoxic injury of islets is a major obstacle for encapsulated islet transplantation into the peritoneal cavity. To improve oxygen delivery to encapsulated islets, we integrated 20% of the oxygen carrier material, perfluorodecalin (PFD), in alginate capsules mixed with islets (PFD-alginate). Integration of PFD clearly improved islet viability and decreased reactive oxygen species production compared to islets encapsulated with alginate only (alginate) and naked islets exposed to hypoxia in vitro. In PFD-alginate capsules, HIF-1α expression was minimal, and insulin expression was well maintained. Furthermore, the best islet function represented by glucose-stimulated insulin secretion was observed for the PFD-alginate capsules in hypoxic condition. For the in vivo study, the same number of naked islets and encapsulated islets (alginate and PFD-alginate) was transplanted into streptozotocin-induced diabetic mice. Nonfasting blood glucose levels and the area under the curve for glucose based on intraperitoneal glucose tolerance tests in the PFD-alginate group were lower than in the alginate group. The harvested islets stained positive for insulin in all groups, but the ratio of dead cell area was 4 times higher in the alginate group than in the PFD-alginate group. In conclusion, integration of PFD in alginate microcapsules improved islet function and survival by minimizing the hypoxic damage of islets after intraperitoneal transplantation.

Anti-Inflammatory Strategies in Intrahepatic Islet Transplantation

The identification of pathway(s) playing a pivotal role in peritransplant detrimental inflammatory events represents the crucial step toward a better management and outcome of pancreatic islet transplanted patients. Recently, we selected the CXCR1/2 inhibition as a relevant strategy in enhancing pancreatic islet survival after transplantation. Here, the most clinically used anti-inflammatory compounds (IL1-receptor antagonist, steroids, and TNF-α inhibitor) alone or in combination with a CXCR1/2 inhibitor were evaluated in their ability to improve engraftment or delay graft rejection. To rule out bias related to transplantation site, we used well-established preclinical syngeneic (250 C57BL/6 equivalent islets in C57BL/6) and allogeneic (400 Balb/c equivalent islets in C57BL6) intrahepatic islet transplantation platforms. In mice, we confirmed that targeting the CXCR1/2 pathway is crucial in preserving islet function and improving engraftment. In the allogeneic setting, CXCR1/2 inhibitor alone could reduce the overall recruitment of transplant-induced leukocytes and significantly prolong the time to graft rejection both as a single agent and in combination with immunosuppression. No other anti-inflammatory compounds tested (IL1-receptor antagonist, steroids, and TNF-α inhibitor) alone or in combination with CXCR1/2 inhibitor improve islet engraftment and significantly delay graft rejection in the presence of MMF + FK-506 immunosuppressive treatment. These findings indicate that only the CXCR1/2-mediated axis plays a crucial role in controlling the islet damage and should be a target for intervention to improve the efficiency of islet transplantation.